Mitochondrial NAD+-specific isocitrate dehydrogenase (IDH) is sensitive to cellular energy levels due to allosteric regulation by adenine nucleotides and NADH. In turn, as a rate-limiting enzyme in the tricarboxylic acid (TCA) cycle, IDH regulates rates of flux through oxidative metabolic pathways. Under conditions of energy sufficiency, this control is fundamental for redirection of carbon and energy flux into biosynthetic metabolic pathways. Yeast IDH is an octamer composed of four homologous regulatory IDH1 and four catalytic IDH2 subunits. IDH1 and IDH2 are 42% identical and contain binding sites that have co-evolved for related regulatory and catalytic ligands. We have determined the crystallographic structure for the octameric yeast enzyme to 2.7 z resolution. The structure demonstrates that the basic structural/functional unit of the enzyme is a heterodimer of IDH1 and IDH2 subunits. We postulate that positive allosteric regulation is controlled at the level of the heterodimer, whereas other layers of organization (of two heterodimers to form the tetramer and of two tetramers to form the octamer) have evolved to restrict activity in the absence of allosteric activation. These and other mechanisms for allosteric regulation of yeast and human IDH enzymes will be investigated using structural approaches (X-ray crystallography) in concert with mutagenesis and with a combination of in vitro and in vivo tests for function. Also, based on recent analyses of cellular metabolite concentrations in idh and other yeast TCA cycle mutants, we formulated and will test new hypotheses that (a) mitochondrial accumulation of citrate contributes to instability of the mitochondrial genome, (b) excess mitochondrial succinate is a signaling molecule for mitochondrial dysfunction and leads to compensatory up regulation of cytosolic synthetic pathways, and (c) major changes in cellular levels and compartmentalization of NAD(H) occur during alterations in environmental conditions. This research seeks to understand the regulation of cellular energy (ATP) production by a key enzyme, NAD+-specific isocitrate dehydrogenase (IDH), in the mitochondrial tricarboxylic acid cycle. Regulation of IDH is essential for normal control of the distribution of cellular resources either into energy production or into biosynthesis, and may also contribute to aberrant patterns of metabolism observed for example in tumors. Our research will also lead to an understanding of how loss or dysfunction of this enzyme can result in mutations and in changes in levels of other metabolites that have important roles in pathogenic conditions and in the process of aging.